Antibodies against type a botulinum neurotoxin

ABSTRACT

Antibodies for binding epitopes of BoNT/A and hybridomas which produce such antibodies are described. The antibodies of the present invention can be used in a method for detecting BoNT/A in a sample and/or in a method for purifying BoNT/A from an impure solution. In addition, the antibodies can be used for passive immunization against BoNT/A intoxication or as intoxication therapy. Another aspect of the invention is a kit for detecting BoNT/A in a sample.

This application claims the benefit of Provisional application No.60/112,632 filed Dec. 17, 1998.

INTRODUCTION

Anaerobic bacterium Clostridium botulinum produces seven immunologicallydistinct but structurally similar botulinum neurotoxins (BoNTs)designated BoNT/A-G that are associated with foodborne, infant, andwound botulism (Montecucco and Schiavo, 1994, Mol. Microbiol. 13, 1-8;Simpson, 1981, Pharmacol. Rev. 33, 155-188). Due to their uniqueproperties, botulinum neurotoxins (BoNTs) have been used to treat avariety of human disorders involving voluntary muscles. BoNT/A wasapproved in 1989 by the Food and Drug Administration for the treatmentof ocular disorders such as strabismus. It has also been used in thetreatment of spasmodic torticollis, limb dystonias, vocal disorders,cerebral palsy, gastrointestinal disorders, and tremors (Schantz andJohnson, 1997, Perspectives in Biology and Medicine 40, 317-327).Interestingly, BoNT has been found to relieve pain and other syndromesassociated with the autonomic nervous system. Injections of BoNT havebeen shown to induce sweating in certain individuals and relievemigraine headache pain in other individuals. The toxin is known todisrupt the mechanism associated with the release of neuronal substancesor neuronal peptides. Besides having the capacity to provide relief froma variety of human diseases, BoNTs also pose a threat as a neurotoxicbiological agents.

Following synthesis, highly active neurotoxin generated by proteolyticcleavage of the CNTs (clostridial neurotoxins). The active neurotoxincomposed of two main chaines that are connected via a disulfide linkage.The location of the enzymatic subunit of the CNTs has been mapped to thesmaller N terminal chain (50-kDa), while the binding and translocationmotifs are located within the larger chain (a 100-kD heavy (H) chain).The 50-kD carboxyl-terminal fragment of the H chain (HC) have beenpostulated to be the receptor binding subunit of the toxin and isthought that this fragment plays a crucial role to direct and pass theenzymatic portion of the CNT accross the vesicle membrane. Thezinc-endopeptidase catalytic domain of the toxin resides in the 50 kDaN-terminal portion of the active protein. Upon entry into the cells BoNTworks at the neurojunction by rendering key neuronal proteins associatedwith the release of acetyl-choline inactive (Montecucco and Schiavo,1994, supra; Blasi et al., 1993, Nature 365, 160-163). Very recently,the structure of BoNT/A was solved and showed substantial homology amongthe structure of the C-terminal binding domain of BoNT/A and Tetanus(Lacy et al., 1998, Nature Struct. Biol. 5, 898-902; Umland et al.,1998, Nature Struct. Biol. 4, 788-792). Unlike the translocation domainof other toxins, the protein fold seen in BoNT/A translocation domaincontains a kinked pair of α-helices and a 54 residue that wraps aroundthe enzymatic domain similar to a “belt” (Lacy and Stevens, 1998, Curr.Opin. Struct. Biol. 8, 778-784). The role that the “belt” plays intoxicity of BoNT has not been studied. However, it may play an importantrole during translocation and cleavage of the substrates.

Although all CNTs are zinc-dependent endopeptidases, they differ insubstrate specificity, substrate cleavage-site location, and their sitesof action within the central nervous system (Lacy et al., 1998, supra).For example, BoNT serotypes A and E cleave SNAP-25(synaptosome-associated protein of 25 kDa), while other CNTs cleavesyntaxin or synaptobrevin. BoNTs inhibit cholinergic vesicle docking atneuromuscular presynaptic nerve endings and cause potentially fatalflaccid paralysis, whereas TeNT (tetanus neurotoxin) is transported in aretrograde manner to the spinal cord, resulting in spastic paralysis anddeath (Montecucco and Schiavo, 1994, supra).

SUMMARY OF THE INVENTION

Probably due to unusually high toxicity of BoNTs, previous attempts toproduce large numbers of high affinity neutralizing monoclonalantibodies (MAbs) against these neurotoxins have been unsuccesful. Wereasoned because immunization with non-toxic binding fragment of BoNT/Acan induce protective immunity in mice, then it should be possible togenerate neutralizing antibodies using this approach. We report hereinthat immunization with BoNT/A-Hc allowed the generation of MAbsrecognizing both the whole BoNT/A and BoNT/A Hc. We characterized theseantibodies in detail, demonstrated biochemical detection of BoNT/A andits binding fragment. We used, neutralizing MAbs directed against theBoNT/A-Hc, in combination with theoretically derived predictions ofsecondary and solvent accessibility of the residues within theBoNT/A-Hc, to locate the principle protective antigenic determinants(PPDS) of BoNT/A-Hc. Binding of the neutralizing MAbs to overlappingtruncated recombinant polypeptides corresponding to BoNT/A-Hc wereexamined. In addition, we tested MAb recognition of two synthetic 25-merpeptides, whose sequences correspond to predicted solvent-exposed loopswithin the C-terminal end of the BoNT/A-Hc. Finally, we examined theability of these peptides to elicit antibody production and to determinewhether the resultant antibodies protected the immunized mice fromBoNT/A challenge. From these experiments, we identified two regionswithin the Hc that may contribute to a neutralizing epitope. Because oftheir ability to neutralize BoNT/A, they could be used for mappingbinding sites of the toxin, for competitive-based ELISA to predictimmunity following vaccination, identify protective epitopes, and theymay be important tools for therapeutic purposes.

Therefore, it is one object of the present invention to provideprotective antibodies against BoNT/A. The antibodies of the presentinvention can be monoclonal or polyclonal antibodies. The presentinvention also pertains to hybridomas producing antibodies, such as4A2-2, 6B2-2, and 6C2-4, which bind to an epitope of BoNT/A.

It is another object of the present invention to provide a method ofpurifying BoNT/A from an impure solution containing BoNT/A. The methodinvolves contacting the impure solution with an antibody which binds anepitope of BoNT/A, allowing the antibody to bind to BoNT/A to form animmunological complex, and separating the complex from the impuresolution. The method of purification can further comprise separating theBoNT/A from the antibody and recovering the BoNT/A. In one embodiment,the separation is conducted by contacting the immunological complex witha saturating amount of peptide comprising the epitope recognized by theantibody of the immunological complex.

The present invention still further pertains to a method for detectingBoNT/A in a sample. The method involves contacting the sample with anantibody which binds an epitope of BoNT/A, allowing the antibody to bindto BoNT/A to form an immunological complex, and detecting the formationof the immunological complex and correlating presence or absence of theimmunological complex with presence or absence of BoNT/A in the sample.The sample can be biological, environmental, or a food sample.

Yet another aspect of the present invention is a kit for detectingBoNT/A in a sample. The kit includes a container holding an antibodywhich binds to an epitope of BoNT/A and instructions for using theantibody for the purpose of binding to BoNT/A to form an immunologicalcomplex and detecting the formation of the immunological complex suchthat presence or absence of the immunological complex correlates withpresence or absence of BoNT/A.

In still another aspect of the invention is provided a method foridentifying the principal protective antigenic deteminant of an antigen.The method includes making neutralizing antibodies using the completeantigen, reacting the neutralizing antibodies to different overlappingfragments encompassing the complete antigen, identifying a fragment ofthe antigen to which most neutralizing antibodies bind, narrowing theregion containing the determinant by reacting the neutralizingantibodies to smaller regions of the identified fragment, andidentifying the regions to which the neutralizing antibodies bind as theprincipal protective antigenic determinant.

In yet another aspect of the invention is provided BoNT/A-Hc peptideswhich have been found to be part of the principal protective antigenicdeterminants identified as SEQ ID NO:1 and SEQ ID NO:2. The peptides canbe used singly or in combination, or the peptide sequences can becombined to produce one sequence and used as one peptide. These peptidesmay be useful as immunogens, and as a vaccine for protecting againtBoNT/A intoxication.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription, appended claims, and accompanying drawings where:

FIG. 1. Neutralizing MAbs recognize denatured BoNT/A Hc. BoNT/A Hc (1mg/lane) was run on denaturing reducing gel and electroblotted onto anitrocellulose membrane. The blotted paper was cut into 12 pieces andeach segment was incubated with each MAb. The bound antibody wasdetected using color substrate. Immunopurified rabbit anti-BoNT/A Hc andSEB-2Ag were positive and negative control, respectively. C-fragmentdenotes BoNT/A Hc.

FIG. 2. The MAbs immunopercipitate BoNT/A Hc. BoNT/A Hc (1 mg/lane) wasincubated with anti-BoNT/A Hc MAbs or anti-SEB (SEB-2Ag) MAb and theMAbs were immunoprecipitated on protein A-Sepharose. The proteins wereseparated on denaturing reducing gel and electroblotted onto anitrocellulose membrane. The proteins were blotted with immunopurifiedrabbit polyclonal anti-BoNT/A Hc and the bound antibody was visualizedusing a Bio-Rad peroxidase development kit. C-fragment denotes BoNT/AHc.

FIG. 3. Affinity mesurement of the neutralizing MAbs. Two representativeof the binding analysis depicted here. Each MAbs was immobilized ontobiosensor chip, after a wash-out phase, different concentrations ofBoNT/A Hc were used to measure K-on and k-off.

FIG. 4. The MAb 6B2-2 binds to a distinct epitope on BoNT/A. Epitopemapping of the MAbs was carried out by SPR, Affinity-purified antibodyto mouse IgG Fc was immobilized onto the chip. Purified-BoNT/A HC MAbwas captured by the antibody, and then nonspecific sites were blocked bypassing a saturating concentration of an unrelated antibody over thematrix surface. BoNT/A HC (200 nM) in HEPES buffered saline was passedover the antibodies at a flow rate of 5 μl/min. Finally, the second(competing) MAb was injected, and its binding determined. The biosensorchip was regenerated and the process was repeated to test the ability ofall MAbs to bind as second MAb using each MAb as first MoAb.

FIG. 5. Neutralizing MAbs recognize two distinct binding sites and oneoverlapping epitope. MAbs 4A2-2, 4A2-4, 6E9-1, 6E9-3, 6E9-4, 6E10-4,6E10-5, 6E10-8, and 6E10-10 recognize the same epitope. These antibodiesrecognize an overlapping epitope as 6C2-4. The MAb 6B2-2 recognizes adistinct epitope.

FIG. 6. Neutralizing MAbs recognize the amino acid residues within thecarboxyl-terminal end of the BoNT/A Hc. The proteins were incubated withanti-BoNT/A Hc MAbs (6E9-12, 4A2-2 or 6C2-2) or anti-SEB (SEB-2Ag) MAband the MAbs were immunoprecipitated on protein A-Sepharose. Theproteins were separated on denaturing reducing gel and electroblottedonto a nitrocellulose membrane. The proteins were blotted with rabbitanti-BoNT/A Hc, peptide corresponding to N-terminal portion of BoNT/A Hcamino acid residues 915-1059 and peptide corresponding to amino acidresidues within the carboxyl-terminal end of the BoNT/A Hc residues1150-1289.

FIG. 7. Location of amino-acid residues corresponding to the designedpeptides. The figure represents the X-ray crystallographic structure ofTeNT-HC on which the positions of amino acids corresponding to peptide 1and peptide 2 are distinguished. Panel (A) shows the alpha-carbonbackbone tracing or, panel (B) depicts the molecular surface of TeNT-HC.

FIG. 8. Binding of protective MAbs to designed peptides. The ability ofMAbs to recognize peptide 1 (A), peptide 2 (B) or control peptide (C)are depicted. The biotinylated-peptides were incubated with MAbs andthen transferred to anti-IgG Fc coated ELISA wells. The boundbiotinylated-peptide in complex with MAb was depicted usingstreptavidin.

FIG. 9. Immunogenicity of the 25-mer designed peptides. Groups of tenBalb/C mice were immunized with peptide 1, peptide 2, BoNT/A Hc orcontrol peptide by i.p. injection (40 mg per mouse for peptides and 5 mgper mouse for BoNT/A Hc). The mice were boosted with the same immunizingdose at 3, 6 and 9 weeks. Two weeks after the last immunization micewere bled and serum titres against BoNT/A Hc determined. Each filledoval represents an endpoint titer of a single animal. Data are presentedas reciprocal serum dilutions resulting in the absorbance reading twiceabove negative control (ELISA wells contained either no BoNT/A Hc or noprimary antibody). The figure shows antibody responses against peptide 1(A), peptide 2 (B), BoNT/A Hc (C) or control peptide (D).

DETAILED DESCRIPTION

The present invention provides antibodies which bind to epitopes ofBoNT/A and BoNT/A Hc. These antibodies can be used to purify BoNT/A froman impure solution containing BoNT/A, and to detect BoNT/A in by asample and as a treatment for BoNT/Z intoxication. In addition, theantibodies can be used in kits for using in the methods described.

The term “antibody” is art-recognized terminology and is intended toinclude molecules or active fragments of molecules that bind to knownantigens. Examples of active fragments of molecules that bind to knownantigens include Fab and F(ab′)₂ fragments. These active fragments canbe derived from an antibody of the present invention by a number oftechniques. For example, purified monoclonal antibodies can be cleavedwith an enzyme, such as pepsin, and subjected to HPLC gel filtration.The appropriate fraction containing Fab fragments can then be collectedand concentrated by membrane filtration and the like. For furtherdescription of general techniques for the isolation of active fragmentsof antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med.23:1011-1019 (1982). The term “antibody” also includes bispecific andchimeric antibodies.

The language “monoclonal antibody” is art-recognized terminology. Themonoclonal antibodies of the present invention can be prepared usingclassical cloning and cell fusion techniques. The immunogen (antigen) ofinterest, e.g. different portions of BoNT/A or BoNT/A Hc, is typicallyadministered (e.g. intraperitoneal injection) to wild-type mice ortransgenic mice which produce desired antibodies, such as humanantibodies, rats, rabbits or other animal species which can producenative or human antibodies. The immunogen can be administered alone oras a fusion protein to induce an immune response. Fusion proteinscomprise the peptide against which an immune response is desired coupledto carrier proteins, such as β-galactosidase, glutathione S-transferase,keyhole limpet hemocyanin (KLH), and bovine serum albumin, to name afew. In these cases, the peptides serve as haptens with the carrierproteins. After the animal is boosted, for example, three or four times,the spleen is removed and splenocytes are extracted and fused withmyeloma cells using the well-known processes of Kohler and Milstein(Nature 256: 495-497 (1975)) and Harlow and Lane (Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory, New York 1988)). Theresulting hybrid cells are then cloned in the conventional manner, e.g.using limiting dilution, and the resulting clones, which produce thedesired monoclonal antibodies, cultured.

Examples of monoclonal antibodies raised against BoNT/A or BoNT/A Hcusing this method include MAb 4A2-2, MAb 6B2-2 and MAb 6C2-4. Themonoclonal antibodies MAb 4A2-2 is produced by the hybridoma depositedunder American Type Culture Collection (ATCC) Accession No. PTA-971 andrecognizes BoNT/A and BoNT/A Hc. MAb 6B2-2 is produced by the hybridomadeposited under American Type Culture Collection (ATCC) Accession No.PTA-969 and recognizes BbNT/A and BoNT/A Hc, and MAb 6C2-4 is producedby the hybridoma deposited under American Type Culture Collection (ATCC)Accession No. PTA-970 recognizes BoNT/A and BoNT/A Hc. Because thesemonoclonals recognize the carboxy terminal of the BoNT/A Hc, a region ofthe Hc found to form an a principal protective antigenic determinant ofBoNT/A, these monoclonals may be useful for passive immunization or forreducing the symptoms of botulinum intoxication. The monoclonals andtheir derivatives can be administered to a subject in an amounteffective to produce protection or reduce symptoms. The amountadministered will depend upon the age, weight and condition of thesubject as described below.

The language “polyclonal antibody” is art-recognized terminology. Theimmunogen used to produce the polyclonals of the present invention wasthe 25 kDa heavy chain of BoNT/A. The polyclonal recognizes BoNT/A.These antibodies are, therefore, useful for studying the topology ofBoNT/A. In addition, these antibodies can be used to determine theorientation of BoNT/A reconstituted into artificial liposomes orvirosomes. The separation of correctly-oriented fromincorrectly-oriented liposomes or virosomes can be achieved usingaffinity chromatography. Anholt et al. J. Biol. Chem. 256:4377 (1981).Because the epitopes recognized by the polyclonal antibody are solventexposed, it is likely a useful antibody for immunoprecipitationexperiments.

A common method for preparing polyclonal antibodies to an immunogen ofinterest, such as BoNT/A or a fragment thereof, includes injecting (e.g.intradermally, intramuscularly) an animal, such as a rabbit, with an theimmunogen emulsified in Freund's complete adjuvant. This process isrepeated after two weeks. Two weeks later, monthly subcutaneous boosterinjections can begin with the immunogen in Freund's incomplete adjuvant.The animals are bled biweekly by a marginal ear vein technique beginningsix weeks after the primary immunization. The collected blood isrefrigerated, allowing clots to form, and the supernatant (antiserum)retained.

The term “epitope” is art-recognized. It is generally understood bythose of skill in the art to refer to the region of an antigen, such asBoNT/A, that interacts with an antibody. An epitope of a peptide orprotein antigen can be formed by contiguous or noncontinguous amino acidsequences of the antigen. BoNT/A, like many large proteins, containsmany epitopes. Examples of BoNT/A epitopes recognized by antibodies ofthe present invention include the amino acid sequences 1150-1289 ofBoNT/A Hc (SEQ ID NO:1), amino acids 1157-1181 (SEQ ID NO:2), and aminoacids 1230-1253 (SEQ ID NO:3). These peptides offer a convenient methodfor eluting BoNT/A bound to either MAb 4A2-2, 4A2-4, 6E9-3, 6E9-4,6E10-4, 6E10-5, 6E10-8, 6E10-10, 6B2-2, and 6C2-4 on immunoaffinitycolumns. For example, when an antibody which recognizes the epitope foreither MAb 4A2-2, MAb 6C2-4, or MAb 6B2-2, is used in an immunoaffinitycolumn to purify BoNT/A, the peptide recognized by the antibody can beadded to the immunoaffinity column to elute the BoNT/A. See below for amore detailed description of the purification of BoNT/A.

Epitope mapping studies described in this application defined threegroups of MAbs, corresponding to two-distinct and one overlappingprotective-epitope regions on BoNT/A Hc. One particular region of theantigen was defined by MAb 6B2-2, while 4A2-2, 4A2-4, 6E10-5, 6E10-8,6E10-10, 6E9-3, 6E9-4, 6E9-12, and 6E10-4 MAbs bound a distinct site.The MAb 6C2-4 defined a site that overlaps with 6E10-5, 6E10-8, 6E10-10,6E9-3, 6E9-4, 6E9-12, and 6E10-4 MAbs. However, 6C2-4 MAb bound anepitope that is distinct from 4A2-2 and 4A2-4 binding site. Thus, basedon this analysis, three possible neutralizing epitopes are recognized bythe MAbs of the present invention.

The epitopes to which the monoclonal antibodies bind on BoNT/A have beenidentified as one of the principal protective antigenic determinants ofBoNT/A, suggesting that an eventual vaccine candidate may includeseveral of these epitopes or peptides containing these antigenicdeterminant. The peptides were selected based on a secondary structureprediction algorithm (Rost and Sander 1994, Protein Struct. Funct. Gen.20, 216-226) that located highly solvent exposed residues of BoNT/A.These highly exposed areas are postulated to be sites of interactionbetween antibodies and BoNT/A. We have identified two peptides describedin SEQ ID NO:2 and SEQ ID NO:3 and based on binding of neutralizing MAbsto these areas, we believe that these two peptides are a part of asingle protective epitope on the Hc of BoNT/A.

By further mapping of the binding site of the monoclonal antibodiesdescribed in this application other peptides useful as a vaccine or atherapeutic can be predicted. Therefore, in another aspect, thisinvention relates to a method for identifying protective antigenicepitopes the method comprising (i) reacting a monoclonal antibodiesdescribed in this application to different overlapping fragmentsencompassing the complete antigen, (ii) identifying a fragment to whichthe neutralizing antibody binds, (iii) narrowing the region containingsites further by reacting the monoclonal with smaller overlappingfragments encompassing the region identified in (ii), and (iv) choosingpeptides to which the antibody binds as possible antigenic epitopes. Thepeptides can then be assayed for their ability to protect an animal fromdisease, or to reduce the severity of disease. Peptides definingantigenic protective epitopes can be used in a vaccine as describedbelow and in the Examples. In addition, the peptides can be used as atherapeutic by competing with BoNT/A for its binding site on the neuron.

A vaccine or therapeutic candidate might comprise these peptidesequences and others. These might be delivered as synthetic peptides, oras fusion proteins, alone or co-administered with cytokines and/oradjuvants or carriers safe for human use, e.g. aluminum hydroxide, toincrease immunogenicity. In addition, sequences such as ubiquitin can beadded to increase antigen processing for more effective immuneresponses. Using the MAbs of the present invention, it is possible tofurther map peptides

The present invention also pertains to hybridomas producing antibodieswhich bind to an epitope of BoNT/A. The term “hybridoma” is artrecognized and is understood by those of ordinary skill in the art torefer to a cell produced by the fusion of an antibody-producing cell andan immortal cell, e.g. a multiple myeloma cell. This hybrid cell iscapable of producing a continuous supply of antibody. See the definitionof “monoclonal antibody” above and the Examples below for a moredetailed description of the method of fusion. The hybridoma whichproduces MAb 4A2-2 is deposited under ATCC Accession Number PTA-971. Thehybridoma which produces MAb 6B2-2 is deposited under ATCC AccessionNumber PTA-969. The hybridoma which produces MAb 6C2-4 is depositedunder ATCC Accession Number PTA-970.

The present invention further pertains to a method for purifying BoNT/Afrom an impure solution containing BoNT/A. The method involvescontacting. the impure solution with an antibody which binds an epitopeof BoNT/A, allowing the antibody to form an immunological complex, andseparating the complex from the impure solution. This method can be usedto clear toxic amounts of BoNT/A from any biological fluid and subjectsincluding animals and humans.

The method of purification can further comprise separating the BoNT/Afrom the antibody and recovering the BoNT/A. In one embodiment, theseparation is conducted by contacting the immunological complex with asaturating amount of peptide comprising the epitope recognized by theantibody of the immunological complex.

The term “purifying” is intended to include removal of unwantedconstituents of an impure solution containing BoNT/A such that theconcentration of BoNT/A in the solution after purification is greaterthat the concentration of BoNT/A in the solution prior to purificationand the concentration of at least one unwanted constituent in thesolution after purification is less than the concentration of theunwanted constituent in the solution prior to purification. Unwantedconstituents include molecules other than BoNT/A. It should beunderstood that the extent of the purification of the solution candepend on the intended use of the BoNT/A. For example, the BoNT/Apurified for therapeutic use will have to be more pure than BoNT/Apurified for research purposes.

The language “impure solution” is intended to include a mixture ofcompounds which includes BoNT/A and at least one non-BoNT/A compound.For example, an impure solution can comprise a biological samplecontaining BoNT/A as defined below.

The language “immunological complex” is intended to include an antigen,such as BoNT/A or a fragment thereof, bound to an antibody, such as amonoclonal or polyclonal antibody, or a fragment thereof. The antigenand antibody are typically bound to one another through noncovalentinteractions.

The immunological complex as well as the antigen alone can be separatedfrom the impure solution by any separation technique known to those ofordinary skill in the art. For example, one commonly used separationmethod is immunoaffinity chromatography. See Harlow and Lane,Antibodies: A Laboratorv Manual (Cold Spring Harbor Laboratory, New York1988) 511-552. Immunoaffinity purification generally consists of threesteps: preparation of an antibody-matrix, binding of an antigen to theantibody-matrix, and elution of the antigen. In the first step, eithermonoclonal antibodies or affinity-purified polyclonal antibodies can becovalently attached to a solid-phase matrix. An example of covalentattachment of the antibody to the solid-phase matrix is linkage of theantibody to protein A beads. See Harlow and Lane, Antibodies: ALaboratorv Manual (Cold Spring Harbor Laboratory, New York 1988)521-523. After the preparation of the antibody-solid phase matrix, theantigen is bound to the antibody and contaminating molecules are removedby washing. In the final step, the antigen-antibody interaction isbroken by treating the immune complexes with strong elution conditions,adding a saturating amount of a small compound (e.g. the peptidecomprising the epitope recognized by the antibody used in the column)that mimics the binding site, and/or treating with an agent whichinduces an allosteric change that releases the antigen, to release theantigen into the eluate. Optimal conditions for binding the antigen tothe column, washing the column to remove contaminants, and eluting thepurified antigen can be determined using conventional parameters as thestarting point and testing the effect of varying the parameters. It isrecognized that effective wash and elution conditions will significantlyimpact the degree of purification obtained. Extensive washing in thepresence of stabilizers plus higher salt and differing detergents can beutilized to remove nonspecifically adsorbed proteins. Elution can thenbe carried out most advantageously by lowering the pH followed byimmediate pH neutralization of the eluted fractions, by using theabove-described specific peptide elution (Courtneige et al., 1984, ColdSpring Harbor Conference on Cell Prolif. and Cancer 2:123), orchaotropic agents such as potassium thiocyanate.

Although it is likely that immunoaffinity chromatography would provide asignificant purification and provide protein of sufficient purity forresearch studies and drug screening, such an approach alone may notprovide adequate protein purity to qualify BoNT/A as a clinical gradetherapeutic agent. Thus, to purify the protein further, or in the casethat immunoaffinity chromatography was unsuccessful, one could test anumber of additional chromatographic approaches to select an optimalchromatography procedure to obtain the desired purity. For example,ligand affinity (Landry et al., 1989 Science 244:1469; Smigel, 1986, J.Biol. Chem. 261:1976), lectin (Curtis and Catterall, 1984, Biochemistry23:2113), anion exchange (Hartshorne and Catterall, 1981, Proc. Natl.Acad. Sci. USA 78:4620) hydroxyapatite (Hartshorne and Catterall, 1984,J. Biol. Chem. 259:1667), and gel filtration (Borsotto et al. 1985, J.Biol. Chem. 260:14255) chromatographic procedures have been used inpurification schemes.

The present invention still further pertains to a method for detectingBoNT/A in a sample containing BoNT/A. The method includes contacting thesample with an antibody which binds an epitope of BoNT/A, allowing theantibody to bind to BoNT/A to form an immunological complex, anddetecting the formation of the immunological complex and correlatingpresence or absence of the immunological complex with presence orabsence of BoNT/A in the sample. The sample can be biological,environmental or a food sample.

The language “detecting the formation of the immunological complex” isintended to include discovery of the presence or absence of BoNT/A in asample. The presence or absence of BoNT/A can be detected using animmunoassay. A number of immunoassays used to detect and/or quantitateantigens are well known to those of ordinary skill in the art. SeeHarlow and Lane, Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory, New York 1988) 555-612. Such immunoassays include antibodycapture assays, antigen capture assays, and two-antibody sandwichassays. These assays are commonly used by those of ordinary skill in theart. In an antibody capture assay, the antigen is attached to solidsupport, and labeled antibody is allowed to bind. After washing, theassay is quantitated by measuring the amount of antibody retained on thesolid support. A variation of this assay is a competitive ELISA whereinthe antigen is bound to the solid support and two solutions containingantibodies which bind the antigen, for example, serum from a BoNT/Avaccinee, and a monoclonal antibody of the present invention, areallowed to compete for binding of the antigen. The amount of monoclonalbound is then measured, and a determination is made whether the serumcontains anti BoNT/A antibodies wherein detection of large amounts ofmonoclonal antibody indicates a small to no antibody against BoNT/A inthe serum. This competitive ELISA can be used to predict immunity in avaccinee following vaccination.

In an antigen capture assay, the antibody is attached to a solidsupport, and labeled antigen is allowed to bind. The unbound proteinsare removed by washing, and the assay is quantitated by measuring theamount of antigen that is bound. In a two-antibody sandwich assay, oneantibody is bound to a solid support, and the antigen is allowed to bindto this first antibody. The assay is quantitated by measuring the amountof a labeled second antibody that can bind to the antigen.

These immunoassays typically rely on labeled antigens, antibodies, orsecondary reagents for detection. These proteins can be labeled withradioactive compounds, enzymes, biotin, or fluorochromes. Of these,radioactive labeling can be used for almost all types of assays and withmost variations. Enzyme-conjugated labels are particularly useful whenradioactivity must be avoided or when quick results are needed.Biotin-coupled reagents usually are detected with labeled streptavidin.Streptavidin binds tightly and quickly to biotin and can be labeled withradioisotopes or enzymes. Fluorochromes, although requiring expensiveequipment for their use, provide a very sensitive method of detection.Antibodies useful in these assays include monoclonal antibodies,polyclonal antibodies, and affinity purified polyclonal antibodies.Those of ordinary skill in the art will know of other suitable labelswhich may be employed in accordance with the present invention. Thebinding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known to those ofordinary skill in the art. Typical techniques are described by Kennedy,J. H., et al., 1976 (Clin. Chim. Acta 70:1-31), and Schurs, A. H. W. M.,et al. 1977 (Clin. Chim Acta 81:1-40). Coupling techniques mentioned inthe latter are the glutaraldehyde method, the periodate method, thedimaleimide method, and others, all of which are incorporated byreference herein.

The language “biological sample” is intended to include biologicalmaterial, e.g. cells, tissues, or biological fluid which, as it existsin nature. By “environmental sample” is meant a sample such as soil andwater. Food samples include canned goods, meats, and others.

Yet another aspect of the present invention is a kit for detectingBoNT/A in a biological sample containing BoNT/A. The kit includes acontainer holding an antibody which binds an epitope of BoNT/A andinstructions for using the antibody for the purpose of binding to BoNT/Ato form an immunological complex and detecting the formation of theimmunological complex such that the presence or absence of theimmunological complex correlates with presence or absence of BoNT/A inthe sample. Examples of containers include multiwell plates which allowsimultaneous detection of BoNT/A in multiple samples.

Treatment of individuals having BoNT/A intoxication may comprise theadministration of a therapeutically effective amount of BoNT/Aantibodies of the present invention. The antibodies can be used as amixture, for example in equal amounts, or individually, provided insequence, or administered all at once. In providing a patient withantibodies, or fragments thereof, capable of binding to BoNT/A, or anantibody capable of inhibiting BoNT/A to a recipient patient, orantibodies to BoNT/A, the dosage of administered agent will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition, previous medical history, etc. In general, itis desirable to provide the recipient with a dosage of antibody which isin the range of from about 1 pg/kg to 10 mg/kg (body weight of patient),although a lower or higher dosage may be administered. When providingthe above-described compounds to a patient, it is preferable toadminister such compounds in a dosage of antibody which is in the rangeof from about 1 pg/kg to 10 mg/kg (body weight of patient), although alower or higher dosage may be administered.

The antibodies capable or inhibiting BoNT/A are intended to be providedto recipient subjects in an amount sufficient to effect a reduction inthe toxicity of BoNT/A. An amount is said to be sufficient to “effect”the reduction of toxicity of BoNT/A if the dosage, route ofadministration, etc. of the agent are sufficient to influence such aresponse. Responses to antibody administration can be measured byanalysis of subject's vital signs.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a “therapeutically effective amount” if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient.

The compounds of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythese materials, or their functional derivatives, are combined inadmixture with a phamaceutically acceptable carrier vehicle. Suitablevehicles and their formulation, inclusive of other human proteins, e.g.,human serum albumin, are described, for example, in Remington'sPharmaceutical Sciences (16th ed., Osol, A. ed., Mack Easton Pa.(1980)). In order to form a pharmaceutically acceptable compositionsuitable for effective administration, such compositions will contain aneffective amount of the above-described compounds together with asuitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb the compounds. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample polyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, metheylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe method of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate the compounds of the present inventioninto particles of a polymeric material such as polyesters, polyaminoacids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, interfacial polymerization, for example,hydroxymethlcellulose or gelatin-microcapsules andpoly(methylmethacylate)-microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

Administration of the antibodies disclosed herein may be carried out byany suitable means, including parenteral injection (such asintraperitoneal, subcutaneous, or intramuscular injection), in ovoinjection of birds, orally, or by topical application of the antibodies(typically carried in a pharmaceutical formulation) to an airwaysurface. Topical application of the antibodies to an airway surface canbe carried out by intranasal administration (e.g., by use of dropper,swab, or inhaler which deposits a pharmaceutical formulationintranasally). Topical application of the antibodies to an airwaysurface can also be carried out by inhalation administration, such as bycreating respirable particles of a pharmaceutical formulation (includingboth solid particles and liquid particles) containing the antibodies asan aerosol suspension, and then causing the subject to inhale therespirable particles. Methods and apparatus for administering respirableparticles of pharmaceutical formulations are well known, and anyconventional technique can be employed. Oral administration may be inthe form of an ingestable liquid or solid formulation.

The treatment may be given in a single dose schedule, or preferably amultiple dose schedule in which a primary course of treatment may bewith 1-10 separate doses, followed by other doses given at subsequenttime intervals required to maintain and or reinforce the response, forexample, at 1-4 months for a second dose, and if needed, a subsequentdose(s) after several months. Examples of suitable treatment schedulesinclude: (i) 0, 1 months and 6 months, (ii) 0, 7 days and 1 month, (iii)0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient toelicit the desired responses expected to reduce disease symptoms, orreduce severity of disease.

The present invention also provides kits which are useful for carryingout the present invention. the present kits comprise a first containermeans containing the above-described antibodies. The kit also comprisesother container means containing solutions necessary or conveninet forcarrying out the invention. The container means can be made of glass,plastic or foil and can be a vial, bottle, pouch, tube, bag, etc. Thekit may also contain written information, such as procedures forcarrying out the present invention or analytical information, such asthe amount of reagent contained in the first container means. Thecontainer means may be in another container means, e.g. a box or a bag,along with the written information.

The contents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof. The following materials and method were used in the examplesbelow.

MATERIALS AND METHODS Antigens

BoNT/A was purchased from List Biochemicals (Campbell, Calif.), andBoNT/A HC , BoNT/B HC, BoNT/E HC were kindly provided by L. Smith(USAMRIID, Frederick, Md.). The BoNT/A HC preparation was analyzed bySDS-PAGE (12%) and stained with Coomassie Brilliant Blue R 250 (SigmaChemical Comp. St Louis, Mo.) in methanol (10% v/v) acidic acid (10%v/v) and found to be at least 95% pure.

The pentavalent toxoid, composed of BoNT serotypes A-E, was obtainedfrom the Bureau of Laboratories, Michigan State Department of PublicHealth (Lansing, Mich.). The five recombinant, overlapping fragmentsencompassing H_(c) of BoNT/A, were purified and generously provided byM. Dertzbaugh (USAMRIID) who previously reported their expression inEscherichia coli (Dertzbaugh and West, 1996, Vaccine 14, 1538-1544). Themolecular masses of these proteins ranged from 18- to 23-kDa. Three25-mer peptides (SEQ ID NO:2 and SEQ ID NO:3), whose sequencescorrespond to regions within BoNT/A, were synthesized, purified, andsequenced by Quality Controlled Biochemicals, Inc. (Hopkinton, Mass.).An additional cysteine residue was added to some of the peptides forbiotinylation procedures.

Experimental Animals

Pathogen-free Balb/C (H-2d) mice, 10- to 12-weeks old, were obtainedfrom Harlan Sprague-Dawley, Inc. (Frederick Cancer Research andDevelopment Center, Frederick, Md.). Mice were maintained underpathogen-free conditions and fed laboratory chow and water ad libitum.

Immunization and Hybridoma Production

Mice were immunized intraperitoneally (i.p.) four times at 4-weekintervals with 2 μg of BoNT/A Hc in 100 μl phosphate-buffered saline(PBS; 50 mM sodium phosphate, 140 mM sodium chloride, pH 7.4). Mice werebled 2 weeks after the last vaccination and titers against BoNT/A andBoNT/A HC were determined. Two mice with the highest titers wereselected for hybridoma production. One month after the last vaccination,these mice were given (ip) 0.1 μg of the antigen and 5 days later,spleens were removed from immunized mice. Splenic mononuclear cells werecollected and fused at a ratio of 3 to 1 with myeloma cells in mediumcontaining polyethylene glycol. After the HAT selection, thesupernatants of the hybridoma cells were tested by ELISA for thepresence of antibodies to the BoNT/A and BoNT/A Hc. Positive hybridomasupernatants were tested for their ability to neutralize BoNT/A, asdescribed below. Limiting dilution was used to clone the hybrids thatproduced neutralizing antibodies, and 33 hybridoma clones secreting MAbsagainst BoNT/A Hc were isolated.

Screening of Hybridoma Supernatants, Antibody Purification,Quantification of MAbs

Either intact BoNT/A or BoNT/A Hc in 100 μl of PBS was coated ontomicrotiter plates at 0.2 μg/well (37° C., 2 h). The wells were blockedwith 250 μl of 0.2% casein in PBS (37° C., 2 h) and then washed fourtimes with wash buffer (PBS containing 0.2% Tween 20, pH 7.4) beforeincubation for 1 h at 37° C. with hybridoma supernatants. The cell freehybridoma supernatants were added undiluted or diluted 1:10 in PBScontaining 0.02% casein. After washing, bound antibody was detected withhorseradish peroxidase (HRP)-labelled goat anti-mouse IgG (Sigma) (37°C., 1 h), with O phenylenediamine as the chromogen. Mean of duplicatesOD (absorbance at 490 nm) of each sample was obtained and scored aspositive if the OD was two times above the negative control wells were.For negative controls, antigen or serum was omitted from the wells

Purification of MAbs was accomplished using Protein G conjugated toSepharose (Pharmacia Biotech, Uppsala, Sweden) according tomanufacturer's instructions.

For quantitation, dilutions of purified MAbs were added to ELISA wellscoated with anti-mouse IgG (Sigma chemical Comp., St. Louis, Mo.). After1 h incubation at 37° C., the captured MAbs were detected using HRPconjugated goat anti-mouse IgG (Cappel/Organon Teknika Corp. , WestChester, Pa.) (37° C., 1 h) and O-phenylenediamine as the chromogen. Themicrotiter-wells were read at 490 nm wells with optical density valuesgreater than two times of the control wells were scored as positive.Standard curves were prepared by plotting the mean of duplicatedensitometric readings for each dilution of IgG standard. The resultingvalues were fitted to a straight line by linear regression.Concentrations of MAbs were determined by comparing mean values ofvarious dilutions of the antibody to the standard curve.

IsotyTe and Subclass Identification

MAb isotype and subclass was determined by standard direct ELISAaccording to manufacturer's instructions (Sigma Chemical Comp.).

Western-immunoblotting and Immunoprecipitation

BoNT/A Hc (1 μg/lane) was separated by SDS-PAGE and transferred tonitrocellulose membranes (Bio-Rad Lab. Inc., Melville, N.Y.) byelectroblotting. Nonspecific sites on the membranes were blocked (12 h4° C.) with 0.2% casein in a buffer consisting of 50 mM sodiumphosphate, 140 mM sodium chloride, pH 7.4 (PBS). The membranes were thenincubated (overnight, room temp, shaking) with 2 μg/mL of affinitypurified anti-BoNT MAb in PBS containing 0.02% casein. After themembranes were thoroughly washed, HRP-conjugated goat anti-mouse IgG(Cappel/Organon Teknika Corp. West Chester, Pa.) was added (1:5,000) andthe membranes were incubated for 1 h (37° C.) with shaking. Unboundantibody was removed by washing with PBS and bound antibody wasvisualized using a Bio-Rad peroxidase development kit.

For immunoprecipitation experiments, nonspecific sites on recombinantprotein A-Sepharose (Repligen Corp., Cambridge, Mass.) were blockedusing 0.2% casein in 10 mM Tris-HCl, pH 7.4 with 150 mM NaCl (TBS) for 2h at 37° C. Unreacted supernatants were collected by centrifugation andthen the precleared protein A-Sepharose (100 μl of 50% suspension) wereincubated (37° C., 2 h, with mixing) with 2 μg of the MAb in 200 μl ofTBS. Immunoadsorbents were collected by centrifugation, washed fourtimes with the same buffer containing 0.02% casein. Bound proteins wereeluted by boiling the samples (95° C., 10 min) in SDS-PAGE buffercontaining 5 mM dithiothreitol. The samples were electrophoresed through12% polyacrylamide gels and analyzed by immunoblotting, as describedabove.

Neutralization Assays

Serial dilutions of MAbs were incubated with various lethal doses ofBoNT/A for 1 h at room temperature. The toxin and antibody mixture wasadministered i.p at a dose of 0.2 ml per mouse. Five days afterchallenge, the mice were scored for survivors. In initial neutralizationassays, mice were observed for up to 20 days.

Binding Kinetics Using Surface Plasmon Resonance

The affinities of MAbs produced to BoNT/A HC were determined with anoptical biosensor using real-time surface plasnion resonance technology(SPR; BIAcore 1000 with upgrade; Biosensor, Pharmacia, San Diego,Calif.). Affinity-purified antibody to mouse IgG Fc and biosensor CM-5chips were purchased from BIAcore AB, (Uppsala, Sweden). The anti-Fcantibody was coupled to the chip with N-hydroxysuccinimide andN-ethyl-N′ (dimethylaminopropyl) carbodiimide, according to themanufacturer's protocol. Each purified BoNT/A HC MAb was captured by thechip immobilized, anti-IgG Fc antibody. Kinetic analyses were carriedout at a flow rate of 25 μl/min with 5-200 nM BoNT/A HC in HEPESbuffered saline. Values for apparent equilibrium dissociation constant(KD) were calculated from the ratio of association (kon) anddissociation (koff) rate constants obtained with the BIAevaluation 2.1software package supplied by the vendor. Biosensor chips with boundanti-IgG Fc antibody were regenerated by removing the bound MAbs withtwo 30-sec pulses of 10 mM glycine (pH 1.8).

Eritope Mapping Using Biosensor Technology

Epitope mapping of the MAbs was carried out by surface plasmon resonanceat a flow rate 5 μl/min. Affinity-purified antibody to mouse IgG Fc wasimmobilized onto the chip, purified BoNT/A HC MAb was captured by theantibody, and then nonspecific sites were blocked by passing asaturating concentration of an unrelated antibody over the matrixsurface. BoNT/A HC (200 nM) in HEPES buffered saline was passed over theantibodies at a flow rate of 5 μl/min. Finally, the second (competing)MAb was injected, and its binding determined. The biosensor chip wasregenerated as above, and the process was repeated to test the abilityof all MAbs to bind as second MAb using each MAb as first MoAb. Thus,all antibody pairs were tested in both directions.

Peptide Immunizations

Ten- to 12-week old mice were immunized (i.p.) with various amounts ofpeptides in 100 μl of RIBI adjuvant (RIBI Immunochem Research, Inc.Hamilton, Mont.), 2-5 μg of BoNT/A/B/E-Hc, or 0.2 ml of pentavalent BoNTtoxoid vaccine. The mice were boosted intraperitoneally (i.p.) at 3 and6 weeks. In some experiments, peptide-immunized mice received a thirdboost at 9 weeks. Serum samples were collected from tail veins 2 weeksafter the last immunization. Mice were challenged 2-3 weeks after thelast injection with intact BoNT/A. Uninjected or adjuvant-injected miceserved as challenge controls.

Antibody Assay

Microtiter plates were coated with 0.2 μg/well of intact BoNT/A, Hc ofBoNT/A, or peptide (1 μg/well) in 100 μl of PBS (37° C., 2 h). Afterantigen coating, the wells were blocked with 250 μl of 0.2% casein inPBS (37° C., 2 h) and then washed four times with PBS containing 0.2%Tween 20. Immune or nonimmune mouse sera were diluted in PBS containing0.02% casein; 100 μl of each dilution was added to duplicate wells.After each well was washed four times, bound antibody was detected withhorseradish peroxidase (HRP)-labelled goat anti-mouse IgG (Sigma) (37°C., 1 h), with O-phenylenediamine as the chromogen. The mean ofduplicate OD (absorbance at 490 nm) of each treatment group was obtainedand these data were compared on the basis of the inverse of the highestserum dilution that produced an OD reading two times greater than thevalue from the negative control wells. For negative controls, antigen orserum was omitted from the wells.

Analysis of MAb Binding to the Designed Deitides

The procedure for peptide-specific ELISA was the same as that describedabove except that microtiter wells were coated with 1 μg of goataffinity-purified antibody produced to mouse IgG Fc (Organon TeknikaCorp. West Chester, Pa.). Non-specific sites were blocked with 250 μl ofcasein 0.2% in PBS (37° C., 2 h) and then washed four times with PBScontaining 0.2% Tween 20. Every MAb was incubated with eachbiotinylated-peptide (37° C., 2 h). The mixture was then added to theanti-IgG Fc-coated wells and incubated at 37° C. for 2 h. Unboundpeptide was washed out of the wells and the bound peptide-MAb complexeswere detected by streptavidin-HRP (SA-HRP; Immunotech, Inc. Westbrook,Me.).

EXAMPLE 1 Generation of Neutralizing MAbs to BoNT/A

We hypothesized that neutralizing antibodies against BoNT/A might bebest generated if one immunizes animals with BoNT/A Hc, the protective,non-toxic receptor-binding. domain of BoNT/A. To generate such MAbs,spleen cells from the highly seropositive mice were fused with myelomacells. To avoid isolation of antibodies that show little to noreactivity against the whole toxin, all of the fusion productsupernatants were screened by a direct ELISA on 96-well platescontaining whole BoNT/A or BoNT/A Hc. This vaccination approach produced660 hybridoma fusions that recognized BoNT/A HC and from these hybridomafusions 488 recognized the native toxin in an ELISA-based assay. Toincrease-the possibilities of finding neutralizing antibodies, weelected to examine protective ability of all 488 fusion-products thatrecognized the toxin in an in vivo mouse lethality model prior tocloning and subsequent subclonning of the hybrids. The cellscorresponding to the positive wells were transferred to 6-well platesand cultured for 7-10 days. The culture supernatants were tested fortheir ability to protect mice against 10 LD50 of BoNT/A. We obtained 33hybrids that produced neutralizing antibody and these fusion-productswere cloned. Candidate hybridomas were subjected to a second round ofexpansion and screening, and were subcloned. Eleven hybridomas werecloned and antibodies were immunopurified using protein G columns. TheMAbs were named: 4A2-2, 4A2-4, 6B2-2, 6C2-4, 6E10-5, 6E10-8, 6E10-10,6E9-3, 6E9-4, 6E9-12, and 6E10-4. When MAbs were tested against 20 LD50of BoNT/A, mice were protected fully (3/3 mice survived). Thespecificity of the MAbs was analyzed by ELISA. The MAbs only recognizedBoNT/A HC and showed no binding to other BoNTs HC and the antibodies didnot offer any protection against other BoNTs (Table 1). These resultsindicate that immunization with the protective fragment of BoNT/Aelicits production of substantial numbers of neutralizing MAbs.

TABLE 1 BoNT/A neutralizing MAbs do not protect against hetrologousBoNTs MAb BoNT serotype LD50 Live/death 4A2-2 A 5 5/0 4A2-2 B 5 0/54A2-2 E 5 0/5 6B2-2 A 5 5/0 6B2-2 B 5 0/5 6B2-2 E 5 0/5 6C2-4 A 5 5/06C2-4 B 5 0/5 6C2-4 E 5 0/5 The antibody was premixed and incubated for1 hr prior to administration to mice.

EXAMPLE 2 Chracterization of the Neutralizing MAbs

To begin to explore the binding characteristics of the antibodies, weexamined in a variety of conditions the interaction of the neutralizingMAbs with the intact BoNT/A and BoNT/A HC including Western immunoblot,and immunoprecipitation. As can be seen in FIG. 1a, the MAbs recognizeda 50 kD protein corresponding to BoNT/A HC. Next we examined the subunitspecificity and epitope of the MAbs were analyzed by Westernimmunoblotting. Purified BoNT/A was boiled in 2-mercaptoethanolcontaining SDS solution, gel electrophoresed, and then Western blottedusing the MAbs. As can be seen in FIG. 1b, BoNT/A degrades into twobands one at approximately 50 kD and the other one at about 100 kD whichthese bands correspond to the light chain and the H chain, respectively.As expected, the MAbs only recognized the H chain. We were alsointerested to evaluate the abilities of the antibodies to detect BoNT/AHC under more physiological conditions. To test this, we studiedimmunoprecipitating soluble BoNT/A HC using the neutralizing antibodiesand then the bound immune complexes was detected followingimmunoblotting with rabbit anti-BoNT/A Hc antibody (FIG. 2). All MAbsimmunoprecipitated the antigen with the same intensity.

To characterize the kinetic interactions between the neutralizing MAbsBoNT/A Hc, we elected to use the SPR (surface plamon resonance)biosensor technology. Each antibody was captured on a biosensor chip,various concentrations of BoNT/A Hc were passed through the flow celland the binding kinetics recorded. Equilibrium dissociation constantsand kinetic on-off rates were measured from the ascending rate ofBIAcore signal during binding and descending rate during wash-offinterval. An example of the SPR studies using MAb 6B2-2 is seen in FIG.3, this figure shows that 6B2-2 possessed an unusually long K_(off)rate. We used SPR and calculated the apparent dissociation constants(KD) of each MAb for BoNT/A HC. Table 2 shows that the majority of theMAbs had similar association rate and their overall values for KD werein the range of 10⁻¹⁰ M. The KD for one of the MAb, 6B2-2, was extremelylower (<5×10⁻¹¹ M) but was difficult to resolve accurately due to itssubstantially slow rate of dissociation, as compared with the otherMAbs. Because the overall affinities were similar for the MAbs examined,we suggest that differences in the binding affinities played only aminor role in the neutralizing ability of the MAbs.

TABLE 2 Kinetic constant of BoNT/A MAbs for BoNT/A Hc k-off k-on KD (nM)4A2-2 3.5 × 10⁻⁴  6.9 × 10⁵ 0.51 4A2-4 4.2 × 10⁻⁴  7.8 × 10⁵ 0.54 6B2-2<0.2 × 10⁻⁴   3.1 × 10⁵ <0.06 6C2-4 1.9 × 10⁻⁴ 17.0 × 10⁵ 0.11 6E9-1 2.5× 10⁻⁴ 11.0 × 10⁵ 0.22 6E9-3 2.8 × 10⁻⁴ 11.0 × 10⁵ 0.25 6E9-4 6.7 × 10⁻⁴ 7.8 × 10⁵ 0.86 6E10-4 5.3 × 10⁻⁴ 11.0 × 10⁵ 0.48 6E10-5 3.9 × 10⁻⁴  4.8× 10⁵ 0.81 6E10-8 2.1 × 10⁻⁴  4.9 × 10⁵ 0.42 6E10-10 3.1 × 10⁻⁴ 14.0 ×10⁵ 0.22 Kinetic constant was measured with SPR.

Next, we used SPR to characterize the binding sites of the Abs. In thesestudies, we used anti-mouse antibody to capture the BoNT/A Hc MAb,followed by BoNT/A Hc. A second competing MAb, was injected, and its wasbinding determined. This experiment was repeated to examine the abilityof all MAbs to bind as second MAb using each MAb as first MAb. This typeof approach tested all pairs of antibodies in both directions. As seenin an example in FIG. 4, when 4A2-2 MAb was immobilized onto the sensorchip and the same antibody or other heterologous antibody, 6B2-2, 6E9-1,and 6E10-8, respectively, were injected sequentially, only competing MAb6B2-2 bound. All combination of antibodies was tested likewise and thedata obtained are summarized in FIG. 5. The epitope mapping studiesdefined three groups of MAbs, corresponding to two-distict and oneoverlapping protective-epitope regions on BoNT/A Hc. One particularregion of the antigen was defeined by MAb 6B2-2, while 4A2-2, 4A2-4,6E10-5, 6E10-8, 6E10-10, 6E9-3, 6E9-4, 6E9-12, and 6E10-4 MAbs bound adistinct site. The MAb 6C2-4defined a site that overlaps with 6E10-5,6E10-8, 6E10-10, 6E9-3, 6E9 4, 6E9-12, and 6E10-4 MoAbs. However, 6C2-4MAb bound an epitope that is distinct from 4A2-2 and 4A2-4 MoAb bindingsite. Thus, based on this analysis, we are proposing that there are atleat two, possibly three, neutralizing epitopes on BoNT/A Hc.

EXAMPLE 3

Immunization with pentavalent toxoid (composed of BoNT serotypes A-E) orH_(c) of BoNT/A produced high antibody titers against BoNT/A andprotected mice against 10, 100, and 1000 LD₅₀ of BoNT/A (Table 3).However, when mice were vaccinated with H_(c) of BoNT serotype B or E,little to no antibody against BoNT/A was detected. These mice were notprotected when challenged with 10 LD₅₀ BoNT/A. This experiment suggeststhere was little to no cross-protection among these serotypes, and thatthe majority of protective epitopes of BoNT/A was located within Hc(Dertzbaugh and West, 1996, supra; Clayton et al., 1995, Infect. Immun.63, 2738-2742).

TABLE 3 Immunization with BoNT/A Hc confers protection Immunization^(a)Titer^(b) Challenge^(c) Live/Dead^(d)(LD50) Toxoid 10⁵ 10 10/0 Toxoid10⁵ 100 10/0 Toxoid 10⁵ 1,000 10/0 BoNT/A Hc 10⁵ 10 10/0 BoNT/A Hc 10⁵100 10/0 BoNT/A Hc 10⁵ 1,000 10/0 BoNT/B Hc <10² 10  1/9 BoNT/E Hc <10²10  0/10 ^(a)Mice were vaccinated i.p. and boosted at 3 and 6 weeks.^(b)Two weeks after the last immunization, mice were bled and serumtiters against BoNT/A determined. Data are presented as reciprocal serumdilution resulting in OD reading twice above negative controls(ELISAwells containing either no BoNT/A or no primary antibody). ^(c)Mice werechallenged with BoNT/A 3 weeks after the final boost. ^(d)Lethality wasrecorded 7 days after the challenge dose.

To identify the sites that contain the neutralizing epitopes, protectiveMAbs to the Hc of BoNT/A were produced in mice.

To characterize the region of BoNT/A H_(c) that these MAbs recognized,the MAbs were reacted separately to each of five recombinant overlappingfragments encompassing Hcof BoNT/A-Hc (Dertzbaugh and West, 1996,supra). As expected, MAbs were able to immunoprecipitate H_(c) of BbNT/A(˜50 kDa) (FIG. 6). The MAbs, 6E9-12, 4A2-2, and 6C2-2,immunoprecipitated the fragment corresponding to residues 1150-1289.However, none of the MAbs recognized the truncated fragmentcorresponding to the N-terminal portion of BoNT/A H_(c) amino acidresidues 915-1059. Although the other fragments were not recognized, orwere only recognized weakly, by the MAbs, they were recognized bypolyclonal antibodies produced to BoNT/A H_(c), which suggested thatthey may contain other B cell epitopes (data not shown). Control MAb(SEB-2Ag) produced to a bacterial superantigen (Ulrich et al., 1995,Trends Microbiol. 3, 463-468), staphylococcal enterotoxin B (SEB), didnot immunoprecipitate any of the fragments. Of the fragments derivedfrom BoNT/A H_(c), only immunization with the 1150-1289 peptide, whichcontained the residues within the carboxyl-terminal end of the BoNT/AH_(c), protected mice from BoNT/A challenge (Dertzbaugh and West, 1996,supra). Together, these data strongly suggest that the majority of PPDsof BoNT/A reside within the carboxyl-terminal end of the molecule.

To localize further the sites of PPDs, three 25-mers were synthesized(Table 4). These peptides were constructed based on a secondarystructure prediction algorithm that located highly solvent-exposedresidues of BoNT/A Hc (Lebeda and Olson, 1994, Prot. Struct. Func. Gen.20, 230-300; Sander et al., 1991, Proteins 9, 56-68; Rost and Sander,1994, Prot. Struct. Func. Gen. 19, 55-72; Lebeda and Olson, 1995,Toxicon 33, 559-567; Lebeda and Olson, 1997, J. Prot. Chem. 16,607-618). The predictions used for this study are considered to behighly accurate based on the recently determined X-ray crystal structureof TeTN Hc (Lebeda and Olson, 1998, J. Prot. Chem.; Umland et al., 1997,supra). As a control, a 25-mer peptide was prepared by using a sequencefrom N-terminal portion of the H-chain. This portion was also predictedto be solvent-exposed. We selected peptides 1 (SEQ ID NO:2) and 2 (SEQID NO:3) because segments of these peptides were predicted to representthe most highly exposed surfaces of BoNT/A Hc (Lebeda and Olson, 1994,supra). The locations of the two peptides, using the tertiary structureof TeNT Hc (Umland et al., 1997, supra), are depicted in FIG. 7.

TABLE 4 Sequence of synthetic 25-mer peptides BoNT/A Peptides Amino acidSequence^(a) SEQ ID NO: Control 449-473 ALNDLCIKVNNWDLFFSPSEDNFTN 4 One1157-1181 GTKFIIKKYASGNKDNIVRNNDRVY 2 Two 1230-1253GITNKCKMNLQDNNGNDIGFIGFHQ 3 ^(a)In some experiments, an additionalcysteine residue was added to N-terminus for biotinylation procedures.

Because recognition of a peptide by neutralizing MAbs is a prerequisitefor the presence of PPADs, we examined binding of the MAbs to engineeredsynthetic peptides. The antibodies were incubated with biotin-labelledpeptides, and then the complexes were captured on the wells of ELISAplates by anti-IgG Fc antibodies. Bound peptides in complex with theneutralizing MAb were detected using streptavidin. The neutralizing MAbsrecognized both peptides that were predicted to be highly exposed (FIGS.8a,b), while the control peptide was not recognized (FIG. 8c). Althoughboth peptides were similarly highly exposed, peptide 2 was betterrecognized than peptide 1 by the MAbs. We failed to detect anyinteraction between the peptides and anti-SEB MAb (SEB-2Ag) (FIG. 8).Because all of the MAbs examined recognized both H_(c)-peptides, it islikely that these spatially proximal peptides correspond to a singleneutralizing epitope on the Hc of BoNT/A.

Because these peptides were recognized by MAbs, this suggested that thepeptides may contain some or all of the PPADs within BoNT/A H_(c), andmay have vaccine efficacy. First, the immunogenicity of the peptides inmice was examined (FIG. 9). In contrast to BoNT/A H_(c), the highestantibody responses to the peptides were elicited only after the fourthdose (FIG. 9). Both peptides 1 and 2 induced a good antibody responsewhen the peptide or BoNT/A H_(c) was used as immunogen in an ELISA. Aswe expected, peptides elicited little cross-reactive antibody responsesto other peptides. Mice immunized with peptide 1 did not develop anantibody response against peptide 2. The same scenario was observed whenmice were immunized with peptide 2 and antibody titers were measuredagainst peptide 1. Immunization with BoNT/A H_(c) yielded high titerantibodies that recognized both peptides; however, there were overallhigher titers against peptide 2. These data suggest that antibodiesagainst these peptides may be naturally produced when mice are immunizedwith H_(c), and that peptide 2, not peptide 1, is the immunodominantantigen. Experiments are in progress to better address this observation.In support of this notion, sera obtained from human volunteers immunizedwith BoNT pentavalent toxoid also recognized synthetic peptidescorresponding to these two regions (Atassi et al., 1996, J. Prot. Chem.15, 691-700). Other experiments suggested that these two peptides mayalso contain T-cell epitopes (data not shown). Mice injected with lowdoses of BoNT/A H_(c) produced high titers, that were much higher thanthose elicited by the peptides. We observed the high titers after thethird immunization. Endpoint antibody titers against H_(c) for miceinjected with BoNT/A H_(c) are shown in FIG. 9c. Control peptideproduced little to no antibody to either peptide or BoNT/A H_(c) (FIG.9d). These data substantiate the fidelity of the synthetic peptides invivo in mimicking the immunogenicity of the naturally processed BoNT/A.

Taken together, the results so far suggested that the epitopes detectedby MAbs were good immunogens and, therefore, the peptides were used ascandidates in a preliminary vaccine trial. Mice were immunized with asingle peptide, combination of peptides, or BoNT/A H_(c), and challenged3 weeks after the final immunization with BoNT/A (Table 5). The highestdose was most effective in eliciting high antibody titers for allpeptides (data not shown). All mice that were vaccinated with 2 or 10 μgof peptide and challenged with 10 LD₅₀ of BoNT/A died. Mice immunizedwith peptide 1 were not protected against any lethal challenge doses.Vaccination with peptide 2 resulted in 40% survival when mice were given10 or 40 μg and challenged with 5 or 10 LD₅₀, respectively. Slightlymore protection was afforded when combination of peptide 1 and 2 wereused in the vaccination protocol. This finding is consistent with thehypothesis that these two peptides may be a part of a single protectiveepitope on the Hc of BoNT/A. All mice vaccinated with BoNT/A H_(c) werecompletely protected against challenge. Mice that received controlpeptide, then challenged with 5 or 10 LD₅₀ of BoNT/A were all killed. Nonoticeable effects on the outcome of these experiments were observedwhen peptides were conjugated to KLH, or administered with otheradjuvants (data not shown).

TABLE 5 Vaccine potential of 25-mer designed peptides Immunizing^(a)Challenge^(c) Live/Total^(d) agent Dose^(b) (μg/mouse) (LD50) Peptide 12 10 0/5 10 10 0/5 40 10 0/5 10 5 0/5 Peptide 2 2 10 0/5 10 10 0/5 40 102/5 10 5 2/5 Peptide 1 + 2 2 10 0/5 10 10 0/5 40 10 3/5 10 5 3/5 BoNT/AHc 2 1,000 5/5 Control Peptide 40 5 0/5 ^(a)Mice were vaccinated i.p.and boosted at 3, 6, and 9 weeks. ^(b)Immunizing dose of each immunogen.^(c)Mice were challenged with BoNT/A 3 weeks after the final boost.^(d)Lethality was recorded 7 days after the challenge dose.

One of the goals of this study was to validate the predictive abilitiesof molecular modelling studies that used theoretically derivedpredictions of secondary structure and solvent accessibility of theresidues to identify surfaces that interact with MAbs. We reasoned thatcareful attention to the kinetics of MAbs may permit us to combine thebenefit of modelling with the high-affinity neutralizing MAbs toidentify selected regions that may play a critical role in formingPPADs. To substantiate further this logic, low-affinity MAbs thatrecognize the peptides or BoNT/A H_(c) are being tested for theirability to protect mice against BoNT/A.

There is some evidence from this study and previously published datathat significant discontinuity may exist in neutralizing epitopes withinBoNT/A (Dertzbaugh and West, 1996, supra). In addition, epitope mappingof these MAbs by using a constrained-peptide display library supportsthe existence of discontinuous epitopes within BoNT/A (M. Segall and S.Bavari, unpublished data). Therefore, we believe a single, short-peptidevaccine may not be feasible for generating protective immunity againstBoNT/A. However, because a single MAb with a very high affinity canblock the lethality of BoNT/A, it might be possible to design a vaccinewith a combination of two peptides.

Unlike BoNT/A H_(c), the highest titers to the peptides were detectedafter the third boost. This may be due to unwanted degradation of thepeptides before reaching receptive major histocompatibility complexclass II molecules. In hope of increasing the immunogenicity ofpeptides, we are currently developing delivery methods to protect betterthe peptides from degradation in acidic compartments ofantigen-presenting cells. To increase efficiency of forming complexeswith major histocompatibility complex class II molecules, peptides arebeing engineered with various signal motifs that should more efficientlydeliver the peptides to peptide-loading compartments ofantigen-presenting cells.

In this targeted survey of two peptides, we have not yet exploreddetails of the kinetics of association and dissociation for peptide andMAbs. However, the initial data suggest there is a correlation betweenthe level of MAb binding to H_(c) of BoNT/A, and the effectiveness ofthe MAb peptide complex formation (D. Pless and S. Bavari, unpublishedobservations). A survey of amino acid substitutions will allow us tomonitor the behavior of the two peptides and identify key amino-acidresidues, particularly at solvent-exposed positions, that could alterbinding to MAbs. This type of approach may further help to boost theefficacy of the peptides.

In conclusion, our study demonstrated that robust molecular modellingstudies that predict secondary structure and locate highlysolvent-exposed residues combined with very high-affinity neutralizingMAbs may be used to identify PPADs. This type of approach can be used asa viable alternative to the expensive and time-consuming methods ofidentifying neutralizing epitopes by synthesizing overwhelming numbersof long peptides.

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What is claimed is:
 1. Monoclonal antibody 6B2-2, produced fromhybridoma cell line having accession number ATCC PTA-969.
 2. Acomposition comprising the monoclonal antibody of claim
 1. 3. Thecontinuous hybridoma cell line having deposit accession number ATCCPTA-969, and clones thereof.
 4. A method for detecting BoNT/A saidmethod comprising: (i) incubating a sample with an effective amount ofone or more monoclonal antibodies with specificity for BoNT/A, saidmonoclonal antibodies comprising 6B2-2 produced by the hybridoma cellline having the accession number ATCC PTA-969, under conditions whichallow the formation of an antibody-BoNT/A complex; and (ii) detectingthe antibody-BoNT/A complex wherein the presence or absence of thecomplex correlates to the presence or absence of BoNT/A in the sample.5. The method for detecting BoNT/A according to claim 4 wherein, saidsample is water, biological, pharmaceutical, or food products.
 6. A kitfor detecting BoNT/A in a biological sample, said kit comprising: (1) acontainer comprising monoclonal antibody 6B2-2 produced by the hybridomacell line having the accession number ATCC PTA-969; and (2) instructionsfor using the antibody for the purpose of binding to BoNT/A to form animmunological complex and detecting said immunological complex such thatthe presence or absence of said immunological complex correlates to thepresence or absence of BoNT/A in said sample.
 7. A method for capturingBoNT/A from a sample, said method comprising contacting said sample withthe monoclonal antibody 6B2-2 produced by the hybridoma cell line havingthe accession number ATCC PTA-969, and isolating the complex formedbetween the BoNT/A in the sample and the monoclonal antibody.
 8. Themethod according to claim 7 wherein said sample is selected from thegroup consisting of: biological fluid and animal tissue.